The present invention is generally in the field of genetically engineered bacterial systems for the enhanced production and recovery of polymers such as intracellular proteins, polyhydroxyalkanoate materials, and extracellular polysaccharides.
Microbial fermentations are used for the manufacture of a large number of pharmaceutical and industrial products, including antibiotics, organic acids, amino acids, proteins, vitamins, polymers such as polyhydroxyalkanoates, and polysaccharides (Atkinson & Mavituna, “Biochemical Engineering and Biotechnology Handbook,” 2nd edition, (Stockton Press, USA 1991)) Increased productivity and recovery of more highly purified product are major areas of development to increase profitability. For many of these products, the industry trend generally is towards higher cell density fermentations to increase productivity. Cell densities in excess of 100 g/L are routinely achieved. Decreasing overall fermentation process costs by increasing the recovery of the product from the cell biomass or, in some cases, from the medium, is another means of increasing productivity.
Polyhydroxyalkanoates (PHAs) are biodegradable and biocompatible thermoplastic materials, produced from renewable resources, with a broad range of industrial and biomedical applications (Williams & Peoples, CHEMTECH 26:38-44 (1996)). A number of bacterial strains and fermentation processes have been described for the production of PHAs, such as described, for example, in Lee & Chang Advances in Biochemical Engineer Biotechnology, 52:27-58 (1995); Poirier et al., Bio/Technology 13:142-50 (1995); Doi, Macromol. Symp. 98:585-99 (1995). PHA production methods using specific bacterial strains are described in U.S. Pat. No. 4,477,654 to Holmes et al., U.S. Pat. No. 5,364,778 to Byrom, and U.S. Pat. No. 5,266,470 to Senior et al. (using Ralstonia eutropha (formerly Alcaligenes eutrophus) from carbohydrates); U.S. Pat. No. 5,346,817 to Akiyama et al. (using Ralstonia eutropha strains grown on fatty acids); U.S. Pat. No. 4,336,334 to Powell et al. (using Methylobacterium organophilum); U.S. Pat. No. 5,302,525 and U.S. Pat. No. 5,434,062 to Groleau et al. (using Methylobacterium extorquens); U.S. Pat. No. 5,292,860 to Shiotani et al. (using Aeromonas strains grown on fatty acids); U.S. Pat. No. 5,059,536 and U.S. Pat. No. 5,096,819 to Page et al. (using Azotobacter vinelandii); U.S. Pat. No. 4,786,598 to Lafferty (using Alcaligenes latus); U.S. Pat. No. 5,344,769 to Witholt et al. and U.S. Pat. No. 5,290,910 to Shiotani et al., PCT WO 92/18553 and PCT WO 92/21708 (using Pseudomonas putida); U.S. Pat. No. 5,245,023 to Peoples et al., U.S. Pat. No. 5,334,520 to Dennis, U.S. Pat. No. 5,371,002 to Dennis et al., U.S. Pat. No. 5,512,456 to Dennis, U.S. Pat. No. 5,518,907 to Dennis et al., and U.S. Pat. No. 5,663,063 to Peoples et al. (using transgenic Escherichia coli).
PHAs accumulate inside the microbial cells as discrete granular inclusions which although amorphous in nature are water insoluble. Recovery of PHAs from the cells following fermentation can be accomplished by any of several methods, including (1) solvent extraction; (2) chemical destruction of all non-PHA biomass using hypochlorite, hydrogen peroxide or ozone treatment; and (3) milder processing involving cell disruption, enzyme treatment, and washing. In the latter process, it is possible to retain the PHA granules in the amorphous state and use the washed granule suspension as a latex. It is necessary to lyse PHA-containing cells in order to obtain a PHA latex from the cells using an aqueous process. Efforts have been made to reduce the viscosity of the lysate. For example, U.S. Pat. No. 4,910,145 to Holmes et al. describes an aqueous process for the extraction of PHA from bacterial biomass, which uses a heat treatment step at 150° C. to reduce the viscosity of the lysate. PCT WO 94/24302 and PCT WO 94/10289 teach the use of hydrogen peroxide treatment to degrade the nucleic acid as a means to reduce viscosity. The addition of nucleases to cell lysates to reduce viscosity and enhance further processing also is generally known. This approach, however, is too expensive to use for commodity fermentation products involving high cell density fermentations.
Fermentation processes are widely used for the manufacture of enzymes and other bioactive proteins. In many cases, recovery can be improved by the addition of nuclease to the crude cell lysates to degrade nucleic acid. Advantageously, this approach also completely degrades DNA, which effectively eliminates the possible spread of antibiotic resistance markers or other genetic elements. As in the case of the PHAs, however, the process of adding exogenous nucleases is expensive.
Microbial polysaccharides are produced by fermentation of a number of different microorganisms. (Delest, P. 1989, pp. 301-313. Fermentation technology of microbial polysaccharides in Gums and Stabilisers for the Food Industry 5, Phillips, G. O., Wedlock, D. J. and Williams, P. A. eds. IRL Press at Oxford University Press, New York). For example, Xanthomonas strains are used commercially for the production of xanthan gum (Kennedy & Bradshaw, Prog. Ind. Microbiol. 19:319-71 (1984)), and have been subjected to genetic engineering techniques (Hassler & Doherty, Biotechnol. Prog. 6:182-87 (1990)). During this and similar fermentations, the viscosity of the medium increases dramatically as the extracellular polysaccharide is produced. Accordingly and in order to achieve good mixing in the fermenter, the mixer requires additional energy. The resulting increased shear can cause some cell lysis to occur, which releases nucleic acid into the medium. This nucleic acid is difficult to remove and presents a significant product quality problem, especially in use of the polysaccharides in biomedical applications, reducing the efficiency of separation processes, such as chromatography, crystallization, precipitation, centrifugation, and filtration.
It is therefore an object of this invention to provide improved methods of production and recovery using fermentation processes, especially those using high cell densities of polyhydroxyalkanoates, polysaccharides, and other intracellular and extracellular components.
It is another object of this invention to provide improved microbial strains for use fermentation processes, particularly those in which cell lysis or high cell densities occurs, or which results in high viscosity.